The aim of this study is to investigate whether Ginkgo biloba extract can augment endothelial progenitor cells numbers, and promote the cells' proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with Ginkgo biloba extract (to make a series of final concentrations: 10 mg/L, 25 mg/L, and 50 mg/L) or vehicle control for the respective time points (6 hours, 12 hours, 24 hours, and 48 h). Endothelial progenitor cells were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR-2, and AC133 with flow cytometry. Endothelial progenitor cells proliferation, migration, and in vitro vasculogenesis activity were assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. Endothelial progenitor cells adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Incubation of isolated human mononuclear cells with Ginkgo biloba extract dose- and time-dependently increased the number of endothelial progenitor cells, maximum at 25 mg/L, 24 hours (approximately 1-fold increase, P < 0.01). In addition, Ginkgo biloba extract also dose- and time-dependently promoted endothelial progenitor cells proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. The results of the present study defined a novel functional effect of Ginkgo biloba extract: the augmentation of endothelial progenitor cells with enhanced functional activity.
Ginkgo biloba extract has been widely used in human therapeutics to treat peripheral arterial occlusive disease and cerebral insufficiency in the elderly as well as coronary artery diseases (CAD). However, the mechanisms of Ginkgo biloba extract are still not very clear. Recently, Ginkgo biloba extract has been reported to have protective effects on endothelial cell injury or endothelial dysfunction induced by many factors. Endothelial dysfunction ultimately loses a balance between the magnitude of injury and the capacity for repair. 1 A variety of evidence suggested that circulating endothelial progenitor cells (EPCs) constituted 1 aspect of this repair process. 1,2 EPCs are a cell population that has the capacity to circulate, proliferate, and differentiate into mature endothelial cells, but which has not yet acquired characteristic mature endothelial markers and has not yet formed a lumen. 3,4 Laboratory evidence suggested that these precursors participated in postnatal neovascularization and reendothelialization. 1–3,5–7 In addition, it has recently been shown that patients at risk for CADs have decreased numbers of circulating EPCs with impaired activity. 8
We hypothesized that Ginkgo biloba extract not only directly protect endothelial cell but also increase EPC numbers and enhance functions at the same time, thus accelerating endothelial repair process, which contributes to protective effects on endothelial cells and improves the clinical symptoms and prognosis of patients with CAD. To test this hypothesis, we measured the numbers and activity of EPCs exposed to Ginkgo biloba extract in this study.
From the Department of Cardiovascular Diseases, Medical School of Zheijiang University, Zheijiang Province, P.R. China.
Received for publication September 29, 2003; accepted October 31, 2003.
Reprints: Dr. XingXiang Wang, Department of Cardiovascular Diseases, Medical School of Zhejiang University, No. 79, Qingchung Road, Hangzhou 310003, Zhejiang Province, P.R. China (e-mail: Wangxx19730312@yahoo.com.cn).